ABSTRACT A critical challenge in understanding the intricate programs underlying development, assembly, function, and dysfunction of the human nervous system is the lack of direct access to intact, functioning human brain tissue for detailed investigation by imaging, recording, and stimulation. My laboratory has been developing methods for directed differentiation of region-specific brain organoids and we have shown that these can combined two by two to study cell-cell interactions and circuit formation in preparations called brain assembloids. However, current in vitro brain models derived from stem cells do not capture neuromodulatory input, which restrict broader applications in neuroscience and modeling of disease. Here, we propose to develop a human 3D cellular platform that includes neuromodulatory projections. More specifically, we will derive from pluripotent stem cells regionalized brain organoids resembling the nucleus raphe in the brainstem and containing serotonergic neurons, and subsequently assemble them with cortical organoids. Using state-of-the-art live imaging, transcriptomics, pharmacology, viral tracing and electrophysiological methods, we plan to study serotonergic input into the cerebral cortex and use this platform to investigate defects in neuronal cross-talk in a severe genetic form of neurodevelopmental disease.